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Title: The isolation, expression and function of the mammalian staufen genes in reproduction
Author: Wood, Timothy R.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2004
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I have isolated several cDNA clones from staufen 1 and staufen 2 from mouse and sheep ovarian cDNA libraries, such that the complete open reading frames of each gene have been determined in both animals. The localisation of these mRNAs has been investigated within mouse and sheep ovary transections by performing in situ hybridisation. The expression of staufen 1 and 2 mRNAs is oocyte specific, with staufen 1 mRNA showing a higher oocyte expression level. A specific antibody was raised against a Staufen 1 fusion protein, isolated by affinity chromatography. Immunohistochemistry (IHC) on mouse an sheep ovary transactions using the antibody showed a granular pattern within the oocyte, with nucleolar staining and a region of the oocyte cytoplasm lacking Staufen 1 granules (RNPs). All of these characteristics suggest translational regulation with possible localisation events occurring. IHC using the antibody on mouse testis transections, revealed a similar granular expression pattern within spermatogonia and spermatocytes that developed in an asymmetric position, similar to acrosomal positioning within primary spermatids.  The expression of Staufen 1 in spermatogenesis again suggests a strong correlation with translational regulation and localisation events. To establish the role of Staufen 1 in reproduction, the transcripts to which it binds needed to be identified. The cDNA sequence of Staufen 1 encoding the interacting RNA binding domains (RBDs) was subcloned into a Yeast tri-Hybrid vector to perform a Staufen 1 – transcript interacting screen using a testes library. The mRNAs that Staufen 1 interacted with included high density Lipoprotein Binding protein (hdLBP or Vigilin), Chaperonin subunit 6 zeta, a transcript similar to sorting nexin 4 and an uncharacterised testis specific transcript. An in situ hybridisation of mouse testes revealed localised Chaperonin subunit and hdLBP mRNA to large globular particles in the same cell type where Staufen 1 protein was expressed. Interestingly, the nucleolar staining of hdLBP in pachytene spermatocytes changes to a single localised position within the primary spermatid cytoplasm, possibly overlapping the position of the acrosome. These results strongly suggest a function involving translational regulation and localisation. The transcripts isolated appear to be involved in RNA and DNA binding, signalling and tubulin recovery, possible linked to microtubule sperm tail production. The receptor of LDL has recently been implicated in Xenopus oocyte polarity, suggesting male and female germ cell development may share some common roles for Staufen 1.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available